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1
Erales, Jenny, Brigitte Gontero, Julian Whitelegge, and Frédéric Halgand. "Mapping of a copper-binding site on the small CP12 chloroplastic protein of Chlamydomonas reinhardtii using top-down mass spectrometry and site-directed mutagenesis." Biochemical Journal 419, no. 1 (March 13, 2009): 75–86. http://dx.doi.org/10.1042/bj20082004.
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CP12 is a small chloroplastic protein involved in the Calvin cycle that was shown to bind copper, a metal ion that is involved in the transition of CP12 from a reduced to an oxidized state. In order to describe CP12's copper-binding properties, copper-IMAC experiments and site-directed mutagenesis based on computational modelling, were coupled with top-down MS [electrospray-ionization MS and MS/MS (tandem MS)]. Immobilized-copper-ion-affinity-chromatographic experiments allowed the primary characterization of the effects of mutation on copper binding. Top-down MS/MS experiments carried out under non-denaturing conditions on wild-type and mutant CP12–Cu2+ complexes then allowed fragment ions specifically binding the copper ion to be determined. Comparison of MS/MS datasets defined three regions involved in metal ion binding: residues Asp16–Asp23, Asp38–Lys50 and Asp70–Glu76, with the two first regions containing selected residues for mutation. These data confirmed that copper ligands involved glutamic acid and aspartic residues, a situation that contrasts with that obtaining for typical protein copper chelators. We propose that copper might play a role in the regulation of the biological activity of CP12.
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McArdle, H. J., S. M. Gross, D. M. Danks, and A. G. Wedd. "Role of albumin's copper binding site in copper uptake by mouse hepatocytes." American Journal of Physiology-Gastrointestinal and Liver Physiology 258, no. 6 (June 1, 1990): G988—G991. http://dx.doi.org/10.1152/ajpgi.1990.258.6.g988.
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It is possible, in vitro, to label albumin with copper either exclusively on the specific binding site or partly on the specific site and also on other sites by altering the pH at which the two ligands are mixed. Copper attached exclusively to the specific site is taken up more rapidly than copper attached to that site and others on albumin. The effect is proportional to the amount of copper on the specific site. Additional histidine stimulates uptake irrespective of the copper binding site on albumin. The effect is related to the histidine on position 3 of the albumin, since it is not seen when dog albumin is labeled under the same conditions. The data suggest that the cell recognizes and presumably binds the copper-albumin (CuAlb) complex but may preferentially recognize the ternary complex formed by CuAlb and histidine. We suggest that, in vivo, copper is bound mainly as the ternary complex and that the structure formed, presumably similar to that formed by a copper-histidine complex, is what is actually recognized by the cell. After binding, the albumin and histidine are released, possibly by a reduction step, and the copper is transported across the membrane. If the copper cannot be transported (as occurs when the cells are incubated at 4 degrees C), it blocks further binding of the ternary complex.
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Cater, Michael A., Sharon La fontaine, and Julian F. B. Mercer. "Copper binding to the N-terminal metal-binding sites or the CPC motif is not essential for copper-induced trafficking of the human Wilson protein (ATP7B)." Biochemical Journal 401, no. 1 (December 11, 2006): 143–53. http://dx.doi.org/10.1042/bj20061055.
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The Wilson protein (ATP7B) is a copper-translocating P-type ATPase that mediates the excretion of excess copper from hep-atocytes into bile. Excess copper causes the protein to traffic from the TGN (trans-Golgi network) to subapical vesicles. Using site-directed mutagenesis, mutations known or predicted to abrogate catalytic activity (copper translocation) were introduced into ATP7B and the effect of these mutations on the intracellular traf-ficking of the protein was investigated. Mutation of the critical aspartic acid residue in the phosphorylation domain (DKTGTIT) blocked copper-induced redistribution of ATP7B from the TGN, whereas mutation of the phosphatase domain [TGE (Thr-Gly-Glu)] trapped ATP7B at cytosolic vesicular compartments. Our findings demonstrate that ATP7B trafficking is regulated with its copper-translocation cycle, with cytosolic vesicular localization associated with the acyl-phosphate intermediate. In addition, mut-ation of the six N-terminal metal-binding sites and/or the trans-membrane CPC (Cys-Pro-Cys) motif did not suppress the consti-tutive vesicular localization of the ATP7B phosphatase domain mutant. These results suggested that copper co-ordination by these sites is not essential for trafficking. Importantly, copper-chelation studies with these mutants clearly demonstrated a requirement for copper in ATP7B trafficking, suggesting the presence of an additional copper-binding site(s) within the protein. The results presented in this report significantly advance our understanding of the regulatory mechanism that links copper-translocation activity with copper-induced intracellular trafficking of ATP7B, which is central to hepatic and hence systemic copper homoeostasis.
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Calabrese, L., and M. Carbonaro. "An e.p.r. study of the non-equivalence of the copper sites of caeruloplasmin." Biochemical Journal 238, no. 1 (August 15, 1986): 291–95. http://dx.doi.org/10.1042/bj2380291.
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The two Type 1 (blue) copper-binding sites of caeruloplasmin were spectroscopically differentiated by the kinetic analysis of the e.p.r. spectra during the redox cycle. One blue copper, with a hyperfine splitting constant (A parallel) of 6.8 mT, which was rapidly reduced, was not reoxidized by oxygen, whereas it was reoxidized by H2O2. The other blue copper (A parallel = 5.8 mT), which was reduced slowly, was rapidly reoxidized by either oxygen or H2O2. A conformational change of the Type 2 copper was concomitant with the fast reduction of Type 1 copper, whereas its reduction occurred during the slow phase. This sequence of events was reversed in the reoxidation step, that is, the Type 2 copper reappeared rapidly as the species with altered conformation and reverted to the symmetry typical of the native state in the slow phase. The specific reaction of a blue-copper site with the H2O2 can tentatively be related to the established ability of caeruloplasmin to prevent ‘oxidative’ attack of proteins and lipids.
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Sinopoli, Alessandro, Antonio Magrì, Danilo Milardi, Matteo Pappalardo, Pietro Pucci, Angela Flagiello, Jeremy J. Titman, et al. "The role of copper(ii) in the aggregation of human amylin." Metallomics 6, no. 10 (2014): 1841–52. http://dx.doi.org/10.1039/c4mt00130c.
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Copper(ii) coordination to human amylin has an influence on the aggregation and cytotoxic features of the polypeptide. Comparative investigations, carried out on a model peptide encompassing the 17–29 aminoacid region of amylin containing the putative metal binding site, support the non-fibrillar nature of the copper(ii) complexes.
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Kekez, Ivana, Mihovil Faletar, Mario Kekez, Laura Cendron, Maya Wright, Giuseppe Zanotti, and Dubravka Matković-Čalogović. "Copper Binding and Oligomerization Studies of the Metal Resistance Determinant CrdA from Helicobacter pylori." Molecules 27, no. 11 (May 24, 2022): 3387. http://dx.doi.org/10.3390/molecules27113387.
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Within this research, the CrdA protein from Helicobacter pylori (HpCrdA), a putative copper-binding protein important for the survival of bacterium, was biophysically characterized in a solution, and its binding affinity toward copper was experimentally determined. Incubation of HpCrdA with Cu(II) ions favors the formation of the monomeric species in the solution. The modeled HpCrdA structure shows a conserved methionine-rich region, a potential binding site for Cu(I), as in the structures of similar copper-binding proteins, CopC and PcoC, from Pseudomonas syringae and from Escherichia coli, respectively. Within the conserved amino acid motif, HpCrdA contains two additional methionines and two glutamic acid residues (MMXEMPGMXXMXEM) in comparison to CopC and PcoCbut lacks the canonical Cu(II) binding site (two His) since the sequence has no His residues. The methionine-rich site is in a flexible loop and can adopt different geometries for the two copper oxidation states. It could bind copper in both oxidation states (I and II), but with different binding affinities, micromolar was found for Cu(II), and less than nanomolar is proposed for Cu(I). Considering that CrdA is a periplasmic protein involved in chaperoning copper export and delivery in the H. pylori cell and that the affinity of the interaction corresponds to a middle or strong metal–protein interaction depending on the copper oxidation state, we conclude that the interaction also occurs in vivo and is physiologically relevant for H. pylori.
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NAKAMURA, Motoyoshi, Tasuku NAKAJIMA, Yasunori OHBA, Seigo YAMAUCHI, Byung Rho LEE, and Eiji ICHISHIMA. "Identification of copper ligands in Aspergillus oryzae tyrosinase by site-directed mutagenesis." Biochemical Journal 350, no. 2 (August 23, 2000): 537–45. http://dx.doi.org/10.1042/bj3500537.
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Copper ligands of the recombinant tyrosinase from the fungus Aspergillus oryzae expressed in Saccharomyces cerevisiae or Escherichia coli were identified by site-directed mutagenesis. The recombinant protyrosinases expressed in S. cerevisiae were assayed for catalytic activities of mono-oxygenase and L-dopa oxidase at pH 5.5 after acid shock at pH 3.0. Replacements of His-63, His-84, His-93, His-290, His-294, His-332 or His-333 with asparagine resulted in mutant enzymes exhibiting no activities. The site-directed mutant Cys82Ala showed that Cys-82 was also an essential residue for the activity. We obtained homogeneous preparations of activated tyrosinases from mutated thioredoxin fusion gene products expressed in E. coli by acid shock. The copper contents of engineered mutants and wild-type enzyme expressed in E. coli were determined by atomic absorption spectrophotometry. The wild-type enzyme contained 2 g-atoms of copper/mol of the subunit. The His63Asn, His84Asn, His93Asn, His290Asn, His294Asn, His332Asn, His333Asn or Cys82Ala substitution decreased copper binding by approx. 50%, indicating that the mutants contain only approx. 1 g-atom of copper/mol of the subunit. The five mutants His63Asn, His93Asn, His290Asn, His294Asn and Cys82Ala contain only one copper ion, which is fully detectable by EPR. From the correlation of g‖ and CuA‖, we deduced that the nitrogen or sulphur donors in the copper ligands should be in a square or a distorted tetrahedral geometric environment. In further atomic absorption spectrophotometry experiments, no copper atom was observed in the seven double mutants His63Asn/His290Asn, His63Asn/His294Asn, His63Asn/His332Asn, His63Asn/His333Asn, Cys82Ala/His290Asn, His84Asn/His333Asn and His93Asn/His290Asn. We propose a new structure of active sites of tyrosinase from A. oryzae: the most likely binding sites of tyrosinase for Cu(A) are His-63, His-84 and His-93, with the remaining conserved Cys-82 providing the fourth ligand. Cu(B) liganded by four histidine residues, His-290, His-294, His-332 and His-333, is identified as new binding motif of Cu(B).
8
Maghool, Shadi, Michael T. Ryan, and Megan J. Maher. "What Role Does COA6 Play in Cytochrome C Oxidase Biogenesis: A Metallochaperone or Thiol Oxidoreductase, or Both?" International Journal of Molecular Sciences 21, no. 19 (September 23, 2020): 6983. http://dx.doi.org/10.3390/ijms21196983.
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Complex IV (cytochrome c oxidase; COX) is the terminal complex of the mitochondrial electron transport chain. Copper is essential for COX assembly, activity, and stability, and is incorporated into the dinuclear CuA and mononuclear CuB sites. Multiple assembly factors play roles in the biogenesis of these sites within COX and the failure of this intricate process, such as through mutations to these factors, disrupts COX assembly and activity. Various studies over the last ten years have revealed that the assembly factor COA6, a small intermembrane space-located protein with a twin CX9C motif, plays a role in the biogenesis of the CuA site. However, how COA6 and its copper binding properties contribute to the assembly of this site has been a controversial area of research. In this review, we summarize our current understanding of the molecular mechanisms by which COA6 participates in COX biogenesis.
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D’Angelo, Paola, Stefano Della Longa, Alessandro Arcovito, Giordano Mancini, Andrea Zitolo, Giovanni Chillemi, Gabriele Giachin, Giuseppe Legname, and Federico Benetti. "Effects of the Pathological Q212P Mutation on Human Prion Protein Non-Octarepeat Copper-Binding Site." Biochemistry 51, no. 31 (July 27, 2012): 6068–79. http://dx.doi.org/10.1021/bi300233n.
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Eakin, Catherine M., Jefferson D. Knight, Charles J. Morgan, Michael A. Gelfand, and Andrew D. Miranker. "Formation of a Copper Specific Binding Site in Non-Native States of β-2-Microglobulin†." Biochemistry 41, no. 34 (August 2002): 10646–56. http://dx.doi.org/10.1021/bi025944a.
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Attar, Narsis, Oscar A. Campos, Maria Vogelauer, Chen Cheng, Yong Xue, Stefan Schmollinger, Lukasz Salwinski, et al. "The histone H3-H4 tetramer is a copper reductase enzyme." Science 369, no. 6499 (July 2, 2020): 59–64. http://dx.doi.org/10.1126/science.aba8740.
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Eukaryotic histone H3-H4 tetramers contain a putative copper (Cu2+) binding site at the H3-H3′ dimerization interface with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We report that the recombinant Xenopus laevis H3-H4 tetramer is an oxidoreductase enzyme that binds Cu2+ and catalyzes its reduction to Cu1+ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu1+ abundance and copper-dependent mitochondrial respiration and Sod1 function in the yeast Saccharomyces cerevisiae. The histone H3-H4 tetramer, therefore, has a role other than chromatin compaction or epigenetic regulation and generates biousable Cu1+ ions in eukaryotes.
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Watmough, Nicholas J., Sarah J. Field, Ross J. L. Hughes, and David J. Richardson. "The bacterial respiratory nitric oxide reductase." Biochemical Society Transactions 37, no. 2 (March 20, 2009): 392–99. http://dx.doi.org/10.1042/bst0370392.
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The two-subunit cytochrome bc complex (NorBC) isolated from membranes of the model denitrifying soil bacterium Paracoccus denitrificans is the best-characterized example of the bacterial respiratory nitric oxide reductases. These are members of the super-family of haem-copper oxidases and are characterized by the elemental composition of their active site, which contains non-haem iron rather than copper, at which the reductive coupling of two molecules of nitric oxide to form nitrous oxide is catalysed. The reaction requires the presence of two substrate molecules at the active site along with the controlled input of two electrons and two protons from the same side of the membrane. In the present paper, we consider progress towards understanding the pathways of electron and proton transfer in NOR and how this information can be integrated with evidence for the likely modes of substrate binding at the active site to propose a revised and experimentally testable reaction mechanism.
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DiSpirito, Alan A., Jeremy D. Semrau, J. Colin Murrell, Warren H. Gallagher, Christopher Dennison, and Stéphane Vuilleumier. "Methanobactin and the Link between Copper and Bacterial Methane Oxidation." Microbiology and Molecular Biology Reviews 80, no. 2 (March 16, 2016): 387–409. http://dx.doi.org/10.1128/mmbr.00058-15.
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SUMMARYMethanobactins (mbs) are low-molecular-mass (<1,200 Da) copper-binding peptides, or chalkophores, produced by many methane-oxidizing bacteria (methanotrophs). These molecules exhibit similarities to certain iron-binding siderophores but are expressed and secreted in response to copper limitation. Structurally, mbs are characterized by a pair of heterocyclic rings with associated thioamide groups that form the copper coordination site. One of the rings is always an oxazolone and the second ring an oxazolone, an imidazolone, or a pyrazinedione moiety. The mb molecule originates from a peptide precursor that undergoes a series of posttranslational modifications, including (i) ring formation, (ii) cleavage of a leader peptide sequence, and (iii) in some cases, addition of a sulfate group. Functionally, mbs represent the extracellular component of a copper acquisition system. Consistent with this role in copper acquisition, mbs have a high affinity for copper ions. Following binding, mbs rapidly reduce Cu2+to Cu1+. In addition to binding copper, mbs will bind most transition metals and near-transition metals and protect the host methanotroph as well as other bacteria from toxic metals. Several other physiological functions have been assigned to mbs, based primarily on their redox and metal-binding properties. In this review, we examine the current state of knowledge of this novel type of metal-binding peptide. We also explore its potential applications, how mbs may alter the bioavailability of multiple metals, and the many roles mbs may play in the physiology of methanotrophs.
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Marx, G., and M. Chevion. "Site-specific modification of albumin by free radicals. Reaction with copper(II) and ascorbate." Biochemical Journal 236, no. 2 (June 1, 1986): 397–400. http://dx.doi.org/10.1042/bj2360397.
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Exposure of albumin to Cu(II) (10-100 microM) and ascorbate (0.1-2 mM) results in extensive molecular modifications, indicated by decreased fluorescence and chain breaks. The rate of utilization of molecular oxygen and ascorbate as a function of Cu(II) concentration is non-linear at copper/albumin ratios of greater than 1. It appears that Cu(II) bound to the tightest albumin-binding site is less available to the ascorbate than the more loosely bound cation. SDS/polyacrylamide-gel electrophoresis reveals new protein bands corresponding to 50, 47, 22, 18 and 3 kDa. For such a cleavage pattern, relatively few (approximately 3) and rather specific chain breaks occurred. Repeated addition of portions of ascorbate to the albumin/Cu(II) mixture results in increased intensity of the new bands. The absence of Cu(II) or the presence of metal chelating agents is inhibitory. There was no evidence of intermolecular cross-linking or of the formation of insoluble, albumin-derived, material. A mechanism is proposed wherein the loosely bound Cu(II) participates in a Fenton-type reaction. This generates OH. radicals, which rapidly inter-react with the protein and modify it in a ‘site-specific’ manner.
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SOLANO, Francisco, Celia JIMÉNEZ-CERVANTES, José H. MARTÍNEZ-LIARTE, José C. GARCÍA-BORRÓN, José R. JARA, and José A. LOZANO. "Molecular mechanism for catalysis by a new zinc-enzyme, dopachrome tautomerase." Biochemical Journal 313, no. 2 (January 15, 1996): 447–53. http://dx.doi.org/10.1042/bj3130447.
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Dopachrome tautomerase (DCT; EC 5.3.3.12) catalyses the conversion of L-dopachrome into 5,6-dihydroxyindole-2-carboxylic acid in the mammalian eumelanogenic biosynthetic pathway. This enzyme, also named TRP2, belongs to a family of three metalloenzymes termed the tyrosinase-related proteins (TRPs). It is well known that tyrosinase has copper in its active site. However, the nature of the metal ion in the active site of DCT is under discussion. Whereas theoretical predictions based on similarity between the protein sequences of the TRPs suggest the presence of copper, the different inhibition pattern of DCT with some metal chelators compared with that of tyrosinase suggests that the nature of the metal ion could differ. Direct estimations of the metal content in purified DCT preparations show the presence of around 1.5 Zn atoms/molecule and the absence of copper. Apoenzyme preparation by treatment of DCT with cyanide or o-phenanthroline followed by reconstitution experiments of tautomerase activity in the presence of different ions confirmed that the metal cofactor for the DCT active site is zinc. Our results are consistent with Zn2+ chelation by the highly conserved histidine residues homologous to the histidines at the classical copper-binding sites in tyrosinase. This finding accounts for the reaction catalysed by DCT, i.e. a tautomerization, versus the copper-mediated oxidations catalysed by tyrosinase. Based on the predicted tetrahedrical co-ordination of the zinc ions in the enzyme active site, a molecular mechanism for the catalysis of L-dopachrome tautomerization is proposed. From the present data, the existence of additional ligands for metal ions other than zinc in the DCT molecule, such as the proposed cysteine iron-binding sites, cannot be completely ruled out. However, if such sites exist, they could be subsidiary binding sites, whose function would be likely to stabilize the protein.
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Percival, S. S., and E. D. Harris. "Regulation of Cu,Zn superoxide dismutase with copper. Caeruloplasmin maintains levels of functional enzyme activity during differentiation of K562 cells." Biochemical Journal 274, no. 1 (February 15, 1991): 153–58. http://dx.doi.org/10.1042/bj2740153.
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K562 cells, a human erythroleukaemic cell line blocked for differentiation, commit towards erythrocytes when exposed to haemin (20 microM). The cells synthesize fetal haemoglobins and show site-specific binding of caeruloplasmin, a plasma copper protein. These events are set into motion by haemin. On the assumption that the binding of caeruloplasmin could reflect a greater need for copper, we sought to determine whether the transfer of 67Cu from caeruloplasmin was accelerated in haemin-induced compared with non-induced K562 cells. Cu,Zn superoxide dismutase (CuZnSOD) was the recipient. Haemin induction caused the K562 cells to lose CuZnSOD activity. By 96 h, the level of SOD activity was less than 60% of that of non-induced cells. The loss was confined entirely to the CuZn form, MnSOD activity staying essentially unchanged. Although CuZnSOD activity declined with the haemin induction, the incorporation of [4,5-3H]lysine into immunoprecipitable CuZnSOD protein was unaffected. There was also no change in CuZnSOD mRNA concentration in haemin-induced cells. Thus a loss of enzyme did not correlate with a decline in the synthesis de novo of CuZnSOD protein. When 48 h-induced cells were transferred to a medium supplemented with 0.2 microM-caeruloplasmin, CuZnSOD activity was restored to control levels in 24 h. Caeruloplasmin also stimulated the incorporation of [3H]lysine into immunoprecipitable CuZnSOD protein. Caeruloplasmin addition may have affected a post-translational regulatory site for CuZnSOD biosynthesis, possibly by providing copper for the newly synthesized enzyme.
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Giangregorio, Nicola, Annamaria Tonazzi, Lara Console, Mario Prejanò, Tiziana Marino, Nino Russo, and Cesare Indiveri. "Effect of Copper on the Mitochondrial Carnitine/Acylcarnitine Carrier Via Interaction with Cys136 and Cys155. Possible Implications in Pathophysiology." Molecules 25, no. 4 (February 13, 2020): 820. http://dx.doi.org/10.3390/molecules25040820.
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The effect of copper on the mitochondrial carnitine/acylcarnitine carrier (CAC) was studied. Transport function was assayed as [3H]carnitine/carnitine antiport in proteoliposomes reconstituted with the native protein extracted from rat liver mitochondria or with the recombinant CAC over-expressed in E. coli. Cu2+ (as well as Cu+) strongly inhibited the native transporter. The inhibition was reversed by GSH (reduced glutathione) or by DTE (dithioerythritol). Dose-response analysis of the inhibition of the native protein was performed from which an IC50 of 1.6 µM for Cu2+ was derived. The mechanism of inhibition was studied by using the recombinant WT or Cys site-directed mutants of CAC. From the dose-response curve of the effect of Cu2+ on the recombinant protein, an IC50 of 0.28 µM was derived. Inhibition kinetics revealed a non-competitive type of inhibition by Cu2+. However, a substrate protection experiment indicated that the interaction of Cu2+ with the protein occurred in the vicinity of the substrate-binding site. Dose-response analysis on Cys mutants led to much higher IC50 values for the mutants C136S or C155S. The highest value was obtained for the C136/155S double mutant, indicating the involvement of both Cys residues in the interaction with Cu2+. Computational analysis performed on the WT CAC and on Cys mutants showed a pattern of the binding energy mostly overlapping the binding affinity derived from the dose-response analysis. All the data concur with bridging of Cu2+ with the two Cys residues, which blocks the conformational changes required for transport cycle.
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Jaenicke, Elmar, Kay Büchler, Jürgen Markl, Heinz Decker, and Thomas R. M. Barends. "Cupredoxin-like domains in haemocyanins." Biochemical Journal 426, no. 3 (February 24, 2010): 373–78. http://dx.doi.org/10.1042/bj20091501.
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Haemocyanins are multimeric oxygen transport proteins, which bind oxygen to type 3 copper sites. Arthropod haemocyanins contain 75-kDa subunits, whereas molluscan haemocyanins contain 350–400-kDa subunits comprising seven or eight different 50 kDa FUs (functional units) designated FU-a to FU-h, each with an active site. FU-h possesses a tail of 100 amino acids not present in the other FUs. In the present study we show by X-ray crystallography that in FU-h of KLH1 (keyhole-limpet-haemocyanin isoform 1) the structure of the tail domain is cupredoxin-like but contains no copper. The copper-free domain 3 in arthropod haemocyanin subunits has also recently been reinterpreted as being cupredoxin-like. We propose that the cupredoxin-like domain in both haemocyanin types once served to upload copper to the active site of the oxygen-binding domain.
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Martins, Lucas Sousa, Jerônimo Lameira, Hendrik G. Kruger, Cláudio Nahum Alves, and José Rogério A. Silva. "Evaluating the Performance of a Non-Bonded Cu2+ Model Including Jahn−Teller Effect into the Binding of Tyrosinase Inhibitors." International Journal of Molecular Sciences 21, no. 13 (July 6, 2020): 4783. http://dx.doi.org/10.3390/ijms21134783.
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Tyrosinase (TYR) is a metalloenzyme classified as a type-3 copper protein, which is involved in the synthesis of melanin through a catalytic process beginning with the conversion of the amino acid l-Tyrosine (l-Tyr) to l-3,4-dihydroxyphenylalanine (l-DOPA). It plays an important role in the mechanism of melanogenesis in various organisms including mammals, plants, and fungi. Herein, we used a combination of computational molecular modeling techniques including molecular dynamic (MD) simulations and the linear interaction energy (LIE) model to evaluate the binding free energy of a set of analogs of kojic acid (KA) in complex with TYR. For the MD simulations, we used a dummy model including the description of the Jahn–Teller effect for Cu2+ ions in the active site of this enzyme. Our results show that the LIE model predicts the TYR binding affinities of the inhibitor in close agreement to experimental results. Overall, we demonstrate that the classical model provides a suitable description of the main interactions between analogs of KA and Cu2+ ions in the active site of TYR.
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Khalil, Abdelouahed, and Tamàs Fülöp. "A comparison of the kinetics of low-density lipoprotein oxidation induced by copper or by γ-rays: Influence of radiation dose-rate and copper concentration." Canadian Journal of Physiology and Pharmacology 79, no. 2 (February 1, 2001): 114–21. http://dx.doi.org/10.1139/y00-080.
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The oxidation of low-density lipoproteins is the first step in the complex process leading to atherosclerosis. The aim of our study was to compare the kinetics of low density lipoprotein oxidation induced by copper ions or by oxygen free radicals generated by60Co γ-rays. The effects of copper concentration and irradiation dose-rate on LDL peroxidation kinetics were also studied. The oxidation of LDL was followed by the measurement of conjugated diene, hydroperoxides, and thiobarbituric acid reactive substance formation as well as α-tocopherol disappearance. In the case of gamma irradiation, the lag-phase before the onset of lipid peroxidation was inversely correlated to the radiation dose-rate. The radiation chemical rates (v) increased with increasing dose-rate. Copper-induced LDL peroxidation followed two kinetic patterns: a slow kinetic for copper concentrations between 520 µM, and a fast kinetic for a copper concentration of 40 µM. The concentration-dependent oxidation kinetics suggest the existence of a saturable copper binding site on apo-B. When compared with γ-rays, copper ions act as drastic and powerful oxidants only at higher concentrations ([Formula: see text]40 µM).Key words: LDL, peroxidation, kinetics, copper, γ-radiolysis, dose-rate.
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Varfolomeeva, Larisa A., Anastasia Yu Solovieva, Nikolai S. Shipkov, Olga G. Kulikova, Natalia I. Dergousova, Tatiana V. Rakitina, Konstantin M. Boyko, Tamara V. Tikhonova, and Vladimir O. Popov. "Probing the Role of a Conserved Phenylalanine in the Active Site of Thiocyanate Dehydrogenase." Crystals 12, no. 12 (December 8, 2022): 1787. http://dx.doi.org/10.3390/cryst12121787.
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Copper-containing enzymes catalyze a broad spectrum of redox reactions. Thiocyanate dehydrogenase (TcDH) from Thioalkalivibrio paradoxus Arh1 enables the bacterium to use thiocyanate as a unique source of energy and nitrogen. Oxidation of thiocyanate takes place in the trinuclear copper center of TcDH with peculiar organization. Despite the TcDH crystal structure being established, a role of some residues in the enzyme active site has yet to be obscured. F436 residue is located in the enzyme active site and conserved among a number of TcDH homologs, however, its role in the copper center formation or the catalytic process is still not clear. To address this question, a mutant form of the enzyme with F436Q substitution (TcDHF436Q) was obtained, biochemically characterized, and its crystal structure was determined. The TcDHF436Q had an unaltered protein fold but did not possess enzymatic activity, whereas it contained all three copper ions, according to ICP-MS data. The structural data showed that the F436Q substitution resulted in a disturbance of hydrophobic interactions within the active site crucial for a correct transition between open/closed forms of the enzyme–substrate channel. Thus, we demonstrated that F436 does not participate in copper ion binding, but rather possesses a structural role in the TcDH active site.
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Baek, Seung-Hun, Angela Hartsock, and James P. Shapleigh. "Agrobacterium tumefaciens C58 Uses ActR and FnrN To Control nirK and nor Expression." Journal of Bacteriology 190, no. 1 (November 2, 2007): 78–86. http://dx.doi.org/10.1128/jb.00792-07.
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ABSTRACT Agrobacterium tumefaciens can grow anaerobically via denitrification. To learn more about how cells regulate production of nitrite and nitric oxide, experiments were carried out to identify proteins involved in regulating expression and activity of nitrite and nitric oxide reductase. Transcription of NnrR, required for expression of these two reductases, was found to be under control of FnrN. Insertional inactivation of the response regulator actR significantly reduced nirK expression and Nir activity but not nnrR expression. Purified ActR bound to the nirK promoter but not the nor or nnrR promoter. A putative ActR binding site was identified in the nirK promoter region using mutational analysis and an in vitro binding assay. A nirK promoter containing mutations preventing the binding of ActR showed delayed expression but eventually reached about 65% of the activity of an equivalent wild-type promoter lacZ fusion. Truncation of the nirK promoter revealed that truncation up to and within the ActR binding site reduced expression, but fragments lacking the ActR binding site and retaining the NnrR binding site showed expression as high as or higher than the full-length fragment. Additional experiments revealed that expression of paz, encoding the copper protein pseudoazurin, was highly reduced in the actR or fnrN mutants and that ActR binds to the paz promoter. Inactivation of paz reduced Nir activity by 55%. These results help explain why Nir activity is very low in the actR mutant even though a nirK promoter with mutations in the ActR binding site showed significant expression.
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Brouwer, M., T. Hoexum-Brouwer, and R. E. Cashon. "A putative glutathione-binding site in CdZn-metallothionein identified by equilibrium binding and molecular-modelling studies." Biochemical Journal 294, no. 1 (August 15, 1993): 219–25. http://dx.doi.org/10.1042/bj2940219.
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Glutathione (GSH) has been found to form a complex with both vertebrate and invertebrate copper-metallothionein (CuMT) [Freedman, Ciriolo and Peisach (1989) J. Biol. Chem. 264, 5598-5605; Brouwer and Brouwer-Hoexum (1991) Arch. Biochem. Biophys. 290, 207-213]. In this paper we report on the interaction of GSH with CdZnMT-I and CdZnMT-II from rabbit liver and with CdMT-I from Blue crab hepatopancreas. Ultrafiltration experiments showed that all three MTs combined with GSH. The measured binding data for the three MTs could be described by a single binding isotherm. The GSH/MT stoichiometry was 1.4 +/- 0.3 and Kdiss. = 14 +/- 6 microM. Partially Zn-depleted MT does not significantly bind GSH, indicating that the GSH-binding site is located on MT's Zn-containing N-terminal domain. The putative GSH-binding site on rabbit liver MT was investigated using molecular-graphics analysis. A cleft on the MT's N-terminal domain, which has the labile Zn-2 at its base, could easily accommodate GSH. Cysteine-ligand exchange between the terminal (non-bridging) Cys-26, bound to Zn-2, and the cysteine in GSH is stereochemically possible. Based on these considerations a model of MT-GSH was built in which GSH's cysteine replaces Cys-26 as a terminal Zn-2 ligand. This complex was energy-minimized by molecular-mechanics calculations, taking into account computed partial electrostatic charges on all atoms, including Cd and Zn. These calculations showed that the MT-GSH complex was thermodynamically more stable than MT, due to favourable non-bonded, electrostatic and van der Waals interactions. Six hydrogen bonds can form between GSH and MT. The average pairwise root-mean-square deviations (RMSD) of the metals in energy-minimized MT and MT-GSH, compared with the metals in the crystal structure, were 0.0087 +/- 0.0028 nm (0.087 +/- 0.028 A) and 0.0168 +/- 0.0087 nm (0.168 +/- 0.087 A) respectively. The RMSD values for the polypeptide-backbone alpha carbons were 0.0136 +/- 0.0060 nm (0.136 +/- 0.060 A) and 0.0491 +/- 0.0380 nm (0.491 +/- 0.380 A) respectively. No other docking sites for GSH were found. The energy-minimized structure of an MT-2-mercaptoethanol complex was somewhat less stable than the native MT domain, attesting to the specificity of the MT-GSH interaction. The possible physiological significance of the MT-GSH interaction is discussed.
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Georgieva, Dessislava Nikolova, Stanka Stoeva, Wolfgang Voelter, and Nicolay Genov. "Viviparus ater Hemocyanin: Investigation of the Dioxygen-Binding Site and Stability of the Oxy- and Apo-Forms." Zeitschrift für Naturforschung C 56, no. 9-10 (October 1, 2001): 843–47. http://dx.doi.org/10.1515/znc-2001-9-1027.
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Abstract The active site of Viviparus ater (mollusc) hemocyanin was investigated using the fact that the binding of dioxygen to the binuclear copper-containing sites of hemocyanins is connected with the appearance of specific dichroic bands which are very sensitive to changes in the structrure and polarity of the environment. Oxy-Viviparus ater hemocyanin exhibits near UV and visible circular dichroism spectra different from those of other molluscan and arthropo-dan hemocyanins. These differences are due probably to variations in the geometry or charge distribution in the dioxygen binding sites of the compared proteins.The thermostability of Viviparus ater hemocyanin and the significance of the copper-dioxy-gen system for the stability were also investigated. “Melting” temperatures, Tm, of 77 °C for the oxy-hemocyanin and 57 °C for the apo-protein were calculated from the denaturation curves which demonstrates the considerable role of the binuclear active site for the thermostability. Viviparus ater hemocyanin is more thermostable than other hemocyanins for which data are published.
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Boyd, Stefanie D., Morgan S. Ullrich, Jenifer S. Calvo, Fatemeh Behnia, Gabriele Meloni, and Duane D. Winkler. "Mutations in Superoxide Dismutase 1 (Sod1) Linked to Familial Amyotrophic Lateral Sclerosis Can Disrupt High-Affinity Zinc-Binding Promoted by the Copper Chaperone for Sod1 (Ccs)." Molecules 25, no. 5 (February 28, 2020): 1086. http://dx.doi.org/10.3390/molecules25051086.
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Zinc (II) ions (hereafter simplified as zinc) are important for the structural and functional activity of many proteins. For Cu, Zn superoxide dismutase (Sod1), zinc stabilizes the native structure of each Sod1 monomer, promotes homo-dimerization and plays an important role in activity by “softening” the active site so that copper cycling between Cu(I) and Cu(II) can rapidly occur. Previously, we have reported that binding of Sod1 by its copper chaperone (Ccs) stabilizes a conformation of Sod1 that promotes site-specific high-affinity zinc binding. While there are a multitude of Sod1 mutations linked to the familial form of amyotrophic lateral sclerosis (fALS), characterizations by multiple research groups have been unable to realize strong commonalities among mutants. Here, we examine a set of fALS-linked Sod1 mutations that have been well-characterized and are known to possess variation in their biophysical characteristics. The zinc affinities of these mutants are evaluated here for the first time and then compared with the previously established value for wild-type Sod1 zinc affinity. Ccs does not have the same ability to promote zinc binding to these mutants as it does for the wild-type version of Sod1. Our data provides a deeper look into how (non)productive Sod1 maturation by Ccs may link a diverse set of fALS-Sod1 mutations.
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Wang, Jiou, Hilda Slunt, Victoria Gonzales, David Fromholt, Michael Coonfield, Neal G. Copeland, Nancy A. Jenkins, and David R. Borchelt. "Copper-binding-site-null SOD1 causes ALS in transgenic mice: aggregates of non-native SOD1 delineate a common feature." Human Molecular Genetics 12, no. 21 (November 1, 2003): 2753–64. http://dx.doi.org/10.1093/hmg/ddg312.
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Wakabayashi, Hironao, Qian Zhou, Keiji Nogami, and Philip J. Fay. "Effects of Single Point Mutations within Proposed Copper Binding Sites on Specific Activity and Inter-Chain Affinity of Factor VIII." Blood 104, no. 11 (November 16, 2004): 1730. http://dx.doi.org/10.1182/blood.v104.11.1730.1730.
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Abstract Copper ions appear important for the structural integrity and the function of factor VIII. Reconstitution studies have shown that Cu2+ increases specific activity of factor VIII as well as increases affinity between heavy chain (HC) and light chain (LC). Based on the ceruloplasmin homology, the existence of three Cu2+ binding sites has been proposed. These include a type 1 site in HC (A1 domain) coordinated by C310, H315, H267, and M320, a type 1 site in LC (A3 domain) coordinated by C2000, H1954, H2005, and M2010, and type 2 site spanning A1 and A3 domains coordinated by H99 and H1957 with possible other additional residues. We rationalized that point mutations within a putative Cu2+ coordination sites would diminish Cu2+ binding at that site, as detected by changes in factor VIII specific activity and/or inter-subunit affinity. We produced several mutant factor VIII proteins bearing point mutation of C310S, H315A, C2000S, H1954A, H99A, or H1957A using a B-domainless human factor VIII vector. Each mutant was stably expressed and purified by SP-sepharose, which bound factor VIII primarily though LC. Western blotting indicated a reduction in the relative amount of HC in C310S and H99A factor VIII forms, suggesting that inter-chain affinity was reduced by these mutations. EDTA-treated factor VIII forms in the presence of Ca2+ were titrated with Cu2+ and activity was monitored using a factor Xa generation assay. The concentration of free Cu2+ was controlled by the presence of EDTA and Ca2+ based on the known values for Cu2+-EDTA and Ca2+-EDTA affinities. The activity regain observed for wild type factor VIII following titration with Cu2+ yielded a Kd = 11.5 ± 2.2 fM. All of the mutants tested retained a high affinity response to Cu2+, suggesting that single point mutations were not sufficient to eliminate Cu2+ binding. However, while Kd values for H1957A (10.3 ± 2.2 fM), H99A (2.1 ± 1.9 fM), H315A (6.7 ± 3.1 fM), and C310S (12.7 ± 3.4 fM) retained similar or slightly reduced affinity for Cu2+, the Kd values for C2000S (304 ± 105 fM) and H1954A (365 ± 105 fM) showed a significant reduction in Cu2+ affinity. When factor VIIIa subunits were titrated with Cu2+ and monitored by intrinsic fluorescence, only purified A1 subunit showed a saturable effect of the signal (Kd = 11.9 ± 4.9 fM), indicating that this subunit contained a high affinity Cu2+ binding site. Taken together, these results suggest that occupancy of the type 1 Cu2+ binding site in A3 contributes to the Cu2+-dependent increase in specific activity of factor VIII and that the type 1 site in A1 and/or the type 2 site contributes to HC-LC association.
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Brauchli, Sven Y., Frederik J. Malzner, Edwin C. Constable, and Catherine E. Housecroft. "Copper(i)-based dye-sensitized solar cells with sterically demanding anchoring ligands: bigger is not always better." RSC Advances 5, no. 60 (2015): 48516–25. http://dx.doi.org/10.1039/c5ra07449e.
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DSC performances of [Cu(N⁁Nanchor)(N⁁Nancillary)]+ dyes with Ph or Me groups adjacent to the copper-binding site in N⁁Nanchor are compared; electrodes with dyes that bleach are regenerated by reimmersion in dye baths containing N⁁Nanchor.
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Faraco, Vincenza, Paola Giardina, and Giovanni Sannia. "Metal-responsive elements in Pleurotus ostreatus laccase gene promoters." Microbiology 149, no. 8 (August 1, 2003): 2155–62. http://dx.doi.org/10.1099/mic.0.26360-0.
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Fungal laccase gene transcription is strongly induced by copper ions; notably, some laccase promoters contain multiple putative metal-responsive elements (MREs). Previously, it has been demonstrated that the Pleurotus ostreatus laccase genes poxc and poxa1b are transcriptionally induced by copper, and several putative MREs were found in the promoter regions of these genes, which extend for about 400 nt upstream of the start codon (ATG). Identification of MRE sequences, which are protected by protein binding in the poxc and poxa1b promoter regions, has been achieved by footprinting analyses. Electromobility shift assays led to the evaluation of the ability of the identified MREs to bind protein(s), and the role of specific nucleotides of these elements in complex formation has also been analysed. The formation of complexes between analysed MREs and fungal proteins requires the absence of metal ions. Proteins extracted from fungus grown in copper-depleted medium are able to form complexes with MREs, whilst proteins extracted from fungus grown in copper-containing medium are able to form complexes only in the presence of a metal chelator. Moreover, copper-depleted proteins are unable to form complexes when copper or zinc ions are added. UV-cross-linking analyses led to the determination of the molecular masses of the MRE-binding proteins. In the poxa1b promoter, a GC-rich region, homologous to the core binding site for transcription factor Sp1, decreases the binding affinity of the adjacent MRE, affecting its interactions with fungal protein factors.
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CATER, Michael A., John FORBES, Sharon La FONTAINE, Diane COX, and Julian F. B. MERCER. "Intracellular trafficking of the human Wilson protein: the role of the six N-terminal metal-binding sites." Biochemical Journal 380, no. 3 (June 15, 2004): 805–13. http://dx.doi.org/10.1042/bj20031804.
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The Wilson protein (ATP7B) is a copper-transporting CPx-type ATPase defective in the copper toxicity disorder Wilson disease. In hepatocytes, ATP7B delivers copper to apo-ceruloplasmin and mediates the excretion of excess copper into bile. These distinct functions require the protein to localize at two different subcellular compartments. At the trans-Golgi network, ATP7B transports copper for incorporation into apo-ceruloplasmin. When intracellular copper levels are increased, ATP7B traffics to post-Golgi vesicles in close proximity to the canalicular membrane to facilitate biliary copper excretion. In the present study, we investigated the role of the six N-terminal MBSs (metal-binding sites) in the trafficking process. Using site-directed mutagenesis, we mutated or deleted various combinations of the MBSs and assessed the effect of these changes on the localization and trafficking of ATP7B. Results show that the MBSs required for trafficking are the same as those previously found essential for the copper transport function. Either MBS 5 or MBS 6 alone was sufficient to support the redistribution of ATP7B to vesicular compartments. The first three N-terminal motifs were not required for copper-dependent intracellular trafficking and could not functionally replace sites 4–6 when placed in the same sequence position. Furthermore, the N-terminal region encompassing MBSs 1–5 (amino acids 64–540) was not essential for trafficking, with only one MBS close to the membrane channel, necessary and sufficient to support trafficking. Our findings were similar to those obtained for the closely related ATP7A protein, suggesting similar mechanisms for trafficking between copper-transporting CPx-type ATPases.
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Jureschi, Monica, Brindusa Alina Petre, Laura Ion, Catalina Ionica Ciobanu, Ion Sandu, and Gabi Drochioiu. "Synthesis of Different Analogs of Ab(9-16) Peptide Mass spectrometric evidence for heavy metal binding." Revista de Chimie 70, no. 9 (October 15, 2019): 3348–53. http://dx.doi.org/10.37358/rc.19.9.7547.
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Amyloid-b (Ab) peptides are proteins associated with Alzheimer�s disease (AD), because the extracellular Ab deposits are the main cause of this disorder. The aggregation of Ab has been shown to depend on the interactions with metal ions, such as copper, zinc, aluminum or iron. The N-terminal sequence of Ab(1-42) or Ab(1-40) peptides, namely Ab(1-16) peptide fragment, is considered the metal binding site involved in AD neurodegeneration and amyloidogenesis. Therefore, we have investigated different peptide sequences to understand the role played by some amino acid residues in metal binding. In this paper, we report the chemical synthesis of Ab(9-16) peptide and its analogs by Fmoc/tBu strategy and the mass spectrometric evidence for metal ion binding to newly synthesized peptides. MALDI-ToF mass spectrometry proved to be a reliable tool to detect and identify the metal ion complexes of all peptides investigated with copper, iron and zinc ions.
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Pirota, Valentina, Enrico Lunghi, Alessandra Benassi, Emmanuele Crespan, Mauro Freccero, and Filippo Doria. "Selective Binding and Redox-Activity on Parallel G-Quadruplexes by Pegylated Naphthalene Diimide-Copper Complexes." Molecules 26, no. 16 (August 19, 2021): 5025. http://dx.doi.org/10.3390/molecules26165025.
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G-quadruplexes (G4s) are higher-order supramolecular structures, biologically important in the regulation of many key processes. Among all, the recent discoveries relating to RNA-G4s, including their potential involvement as antiviral targets against COVID-19, have triggered the ever-increasing need to develop selective molecules able to interact with parallel G4s. Naphthalene diimides (NDIs) are widely exploited as G4 ligands, being able to induce and strongly stabilize these structures. Sometimes, a reversible NDI-G4 interaction is also associated with an irreversible one, due to the cleavage and/or modification of G4s by functional-NDIs. This is the case of NDI-Cu-DETA, a copper(II) complex able to cleave G4s in the closest proximity to the target binding site. Herein, we present two original Cu(II)-NDI complexes, inspired by NDI-Cu-DETA, differently functionalized with 2-(2-aminoethoxy)ethanol side-chains, to selectively drive redox-catalyzed activity towards parallel G4s. The selective interaction toward parallel G4 topology, controlled by the presence of 2-(2-aminoethoxy)ethanol side chains, was already firmly demonstrated by us using core-extended NDIs. In the present study, the presence of protonable moieties and the copper(II) cavity, increases the binding affinity and specificity of these two NDIs for a telomeric RNA-G4. Once defined the copper coordination relationship and binding constants by competition titrations, ability in G4 stabilization, and ROS-induced cleavage were analyzed. The propensity in the stabilization of parallel topology was highlighted for both of the new compounds HP2Cu and PE2Cu. The results obtained are particularly promising, paving the way for the development of new selective functional ligands for binding and destructuring parallel G4s.
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Pfeuffer, I., S. Klein-Hessling, A. Heinfling, S. Chuvpilo, C. Escher, T. Brabletz, B. Hentsch, H. Schwarzenbach, P. Matthias, and E. Serfling. "Octamer factors exert a dual effect on the IL-2 and IL-4 promoters." Journal of Immunology 153, no. 12 (December 15, 1994): 5572–85. http://dx.doi.org/10.4049/jimmunol.153.12.5572.
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Abstract The promoters of IL-2 and IL-4 genes contain multiple binding sites for octamer factors. In peripheral T lymphocytes and several T cell lines, both the ubiquitous Oct factor Oct-1 and the lymphocyte-specific factor Oct-2 are expressed and bind to the IL-2 and IL-4 promoters. Prominent octamer binding sites of IL-2 and IL-4 promoters are their upstream promoter sites (UPS) which share 14 identical nucleotides. Multiple copies of the IL-2 and IL-4 UPS act as inducible enhancers in T cells, and their induction is inhibited by the immunosuppressant cyclosporin A (CsA). Closely linked to the octamer site, the IL-2 UPS contains a non-canonical AP-1 binding (TRE) site, and mutation in either site to a non-functional factor binding site impairs the induction of the IL-2 promoter. The binding of AP-1 and octamer factors to the IL-2 UPS DNA overlaps, and the tight association and functional cooperation of octamer with AP-1 factors is of crucial importance for the inducible IL-2 UPS activity. Introduction of five or ten spacer nucleotides between both IL-2 UPS sites results in a drastic reduction of inducible UPS activity, both in the loss of suppression by CsA and stimulation by the Ca(2+)-dependent phosphatase calcineurin. Within the IL-4 UPS the Oct and TRE-like motifs are separated by a binding site of nuclear factor of activated T cells (NF-AT). This site shares nine out of ten bp with an IL-2 NF-AT site. The strong binding of NF-ATp to the IL-4 UPS site suppresses the simultaneous binding of Oct factors to the IL-4 UPS. Because the two other Oct binding sites of IL-4 promoter show a similar sequence configuration, the binding of NF-AT seems to prevent the simultaneous binding of Oct factors to the IL-4 promoter. By contrast, both classes of factors bind simultaneously to the IL-2 promoter, and their tight association with AP-1 enhances the IL-2 promoter activity.
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ABRAHAM, Zelda H. L., Barry E. SMITH, Barry D. HOWES, David J. LOWE, and Robert R. EADY. "pH-dependence for binding a single nitrite ion to each type-2 copper centre in the copper-containing nitrite reductase of Alcaligenes xylosoxidans." Biochemical Journal 324, no. 2 (June 1, 1997): 511–16. http://dx.doi.org/10.1042/bj3240511.
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The first quantitative characterization of the interaction of NO2- with the Cu-containing dissimilatory nitrite reductase (NiR) of Alcaligenes xylosoxidansusing steady-state kinetics, equilibrium gel filtration and EPR spectroscopy is described. Each molecule of this protein consists of three equivalent subunits, each containing a type-1 Cu atom and also a type-2 Cu atom at each subunit interface. Enzyme activity increased in a biphasic manner with decreasing pH, having an optimum at pH 5.2 and a plateau between pH 6.1 and 5.8. Equilibrium gel filtration showed that binding of NO2- to the oxidized NiR was also pH-dependent. At pH 7.5, no binding was detectable, but binding was detectable at lower pH values. At pH 5.2, the concentration-dependence for binding of NO2- to the enzyme showed that approx. 4.1 NO2- ions bound per trimeric NiR molecule. Unexpectedly, NiR deficient in type-2 Cu centres bound 1.3 NO2- ions per trimer. When corrected for this binding, a value of 3 NO2- ions bound per trimer of NiR, equivalent to the type-2 Cu content. The NO2--induced changes in the EPR parameters of the type-2 Cu centre of the oxidized enzyme showed a similar pH-dependence to that of the activity. Binding constants for NO2- at a single type of site, after allowing for the non-specifically bound NO2-, were 350±35 μM (mean±S.E.M.) at pH 7.5 and <30 μM at pH 5.2. The apparent Km for NO2- with saturating concentrations of dithionite as reductant was 35 μM at pH 7.5, which is 10-fold tighter than for the oxidized enzyme, and is compatible with an ordered mechanism in which the enzyme is reduced before NO2- binds.
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Shcheglovitov, Aleksandr, Iuliia Vitko, Roman M. Lazarenko, Peihan Orestes, Slobodan M. Todorovic, and Edward Perez-Reyes. "Molecular and biophysical basis of glutamate and trace metal modulation of voltage-gated Cav2.3 calcium channels." Journal of General Physiology 139, no. 3 (February 27, 2012): 219–34. http://dx.doi.org/10.1085/jgp.201110699.
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Here, we describe a new mechanism by which glutamate (Glu) and trace metals reciprocally modulate activity of the Cav2.3 channel by profoundly shifting its voltage-dependent gating. We show that zinc and copper, at physiologically relevant concentrations, occupy an extracellular binding site on the surface of Cav2.3 and hold the threshold for activation of these channels in a depolarized voltage range. Abolishing this binding by chelation or the substitution of key amino acid residues in IS1–IS2 (H111) and IS2–IS3 (H179 and H183) loops potentiates Cav2.3 by shifting the voltage dependence of activation toward more negative membrane potentials. We demonstrate that copper regulates the voltage dependence of Cav2.3 by affecting gating charge movements. Thus, in the presence of copper, gating charges transition into the “ON” position slower, delaying activation and reducing the voltage sensitivity of the channel. Overall, our results suggest a new mechanism by which Glu and trace metals transiently modulate voltage-dependent gating of Cav2.3, potentially affecting synaptic transmission and plasticity in the brain.
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Naro, Fabio, Maria G. Tordi, Giorgio M. Giacometti, Francesco Tomei, Anna M. Timperio, and Lello Zolla. "Metal Binding to Pseudomonas aeruginosa Azurin: a Kinetic Investigation." Zeitschrift für Naturforschung C 55, no. 5-6 (June 1, 2000): 347–54. http://dx.doi.org/10.1515/znc-2000-5-609.
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The interaction between azurin from Pseudomonas aeruginosa and Ag(I), Cu(II), Hg(II), was investigated as a function of protein state, i.e. apo-, reduced and oxidised azurin. Two different metal binding sites, characterized by two different spectroscopic absorbancies, were detected: one is accessible to Ag(I) and Cu(II) but not to Hg(II); the other one binds Ag(I) and Hg(II) but not copper. When added in stoichiometric amount, Ag(I) shows high affinity for the redox center of apo-azurin, to which it probably binds by the -SH group of Cys112; it can displace Cu(I) from reducedazurin, while it does not bind to the redox center of oxidizedazurin. Kinetic experiments show that Ag(I) binding to the reducedform is four times faster than binding to the apo-form. This result suggests that metal binding requires a conformational rearrangement of the active site of the azurin. Interaction of A g(I) or Hg(II) ions to the second metal binding site, induces typical changes of UV spectrum and quenching of fluorescence emission.
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Teodori, Laura, Marjan Omer, Anders Märcher, Mads K. Skaanning, Veronica L. Andersen, Jesper S. Nielsen, Emil Oldenburg, Yuchen Lin, Kurt V. Gothelf, and Jørgen Kjems. "Site-specific nanobody-oligonucleotide conjugation for super-resolution imaging." Journal of Biological Methods 9, no. 1 (March 1, 2022): e159. http://dx.doi.org/10.14440/jbm.2022.381.
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Camelid single-domain antibody fragments, also called nanobodies, constitute a class of binders that are small in size (~15 kDa) and possess antigen-binding properties similar to their antibody counterparts. Facile production of recombinant nanobodies in several microorganisms has made this class of binders attractive within the field of molecular imaging. Particularly, their use in super-resolution microscopy has improved the spatial resolution of molecular targets due to a smaller linkage error. In single-molecule localization microscopy techniques, the effective spatial resolution can be further enhanced by site-specific fluorescent labeling of nanobodies owing to a more homogeneous protein-to-fluorophore stoichiometry, reduced background staining and a known distance between dye and epitope. Here, we present a protocol for site-specific bioconjugation of DNA oligonucleotides to three distinct nanobodies expressed with an N- or C-terminal unnatural amino acid, 4-azido-L-phenylalanine (pAzF). Using copper-free click chemistry, the nanobody-oligonucleotide conjugation reactions were efficient and yielded highly pure bioconjugates. Target binding was retained in the bioconjugates, as demonstrated by bio-layer interferometry binding assays and the super-resolution microscopy technique, DNA points accumulation for imaging in nanoscale topography (PAINT). This method for site-specific protein-oligonucleotide conjugation can be further extended for applications within drug delivery and molecular targeting where site-specificity and stoichiometric control are required.
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Scala, David J., and Lee J. Kerkhof. "Diversity of Nitrous Oxide Reductase (nosZ) Genes in Continental Shelf Sediments." Applied and Environmental Microbiology 65, no. 4 (April 1, 1999): 1681–87. http://dx.doi.org/10.1128/aem.65.4.1681-1687.1999.
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ABSTRACT Diversity of the nitrous oxide reductase (nosZ) gene was examined in sediments obtained from the Atlantic Ocean and Pacific Ocean continental shelves. Approximately 1,100 bp of thenosZ gene were amplified via PCR, using nosZgene-specific primers. Thirty-seven unique copies of thenosZ gene from these marine environments were characterized, increasing the nosZ sequence database fourfold. The average DNA similarity for comparisons between all 49 variants of the nosZ gene was 64% ± 10%. Alignment of the derived amino acid sequences confirmed the conservation of important structural motifs. A highly conserved region is proposed as the copper binding, catalytic site (CuZ) of the mature protein. Phylogenetic analysis demonstrated three major clusters ofnosZ genes, with little overlap between environmental and culture-based groups. Finally, the two non-culture-based gene clusters generally corresponded to sampling location, implying that denitrifier communities may be restricted geographically.
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Abbas, Ioana M., Marija Vranic, Holger Hoffmann, Ahmed H. El-Khatib, María Montes-Bayón, Heiko M. Möller, and Michael G. Weller. "Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR." International Journal of Molecular Sciences 19, no. 8 (August 2, 2018): 2271. http://dx.doi.org/10.3390/ijms19082271.
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Hepcidin-25 was identified as the main iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II)-binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandem mass spectrometry (MS/MS), high-resolution mass spectrometry (HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D) model of hepcidin-25 with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or reference material comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.
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Sen, Kakali, Sam Horrell, Demet Kekilli, Chin W. Yong, Thomas W. Keal, Hakan Atakisi, David W. Moreau, Robert E. Thorne, Michael A. Hough, and Richard W. Strange. "Active-site protein dynamics and solvent accessibility in nativeAchromobacter cycloclastescopper nitrite reductase." IUCrJ 4, no. 4 (June 16, 2017): 495–505. http://dx.doi.org/10.1107/s2052252517007527.
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Microbial nitrite reductases are denitrifying enzymes that are a major component of the global nitrogen cycle. Multiple structures measured from one crystal (MSOX data) of copper nitrite reductase at 240 K, together with molecular-dynamics simulations, have revealed protein dynamics at the type 2 copper site that are significant for its catalytic properties and for the entry and exit of solvent or ligands to and from the active site. Molecular-dynamics simulations were performed using different protonation states of the key catalytic residues (AspCATand HisCAT) involved in the nitrite-reduction mechanism of this enzyme. Taken together, the crystal structures and simulations show that the AspCATprotonation state strongly influences the active-site solvent accessibility, while the dynamics of the active-site `capping residue' (IleCAT), a determinant of ligand binding, are influenced both by temperature and by the protonation state of AspCAT. A previously unobserved conformation of IleCATis seen in the elevated temperature series compared with 100 K structures. DFT calculations also show that the loss of a bound water ligand at the active site during the MSOX series is consistent with reduction of the type 2 Cu atom.
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Goto, Yoshio, and Judith P. Klinman. "Binding of Dioxygen to Non-Metal Sites in Proteins: Exploration of the Importance of Binding Site Size versus Hydrophobicity in the Copper Amine Oxidase fromHansenula polymorpha†." Biochemistry 41, no. 46 (November 2002): 13637–43. http://dx.doi.org/10.1021/bi0204591.
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Frank, L. H., H. K. Cheung, and R. S. Cohen. "Identification and characterization of Drosophila female germ line transcriptional control elements." Development 114, no. 2 (February 1, 1992): 481–91. http://dx.doi.org/10.1242/dev.114.2.481.
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The highly organized structure of the Drosophila ovary makes it an ideal system for studying mechanisms of differential gene expression. Here we report the identification of a 171 bp sequence from the 5' end of the hsp26 gene that functions as a female germ-line-specific transcriptional regulator when linked in two copies to a basal promoter. The regulator is active only in nondividing cells of the germ line, i.e., only in nurse cells and oocytes. It is not active in any examined tissue or cell type outside of the female germ line. Copper nuclease footprinting studies show that the germ line regulator contains two binding sites for each of two different ovarian nuclear factors. Point mutations in the DNA target sites of either nuclear factor abolish in vitro binding and in vivo transcriptional activity, indicating that each factor is a positive activator of nurse cell/oocyte transcription. The two factors may represent different classes of activator proteins, since an increase in the copy number of one factor's DNA target site cannot compensate for a decrease in the copy number of the other factor's target site.
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Maniak, Halina, Michał Talma, Konrad Matyja, Anna Trusek, and Mirosław Giurg. "Synthesis and Structure-Activity Relationship Studies of Hydrazide-Hydrazones as Inhibitors of Laccase from Trametes versicolor." Molecules 25, no. 5 (March 10, 2020): 1255. http://dx.doi.org/10.3390/molecules25051255.
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A series of hydrazide-hydrazones 1–3, the imine derivatives of hydrazides and aldehydes bearing benzene rings, were screened as inhibitors of laccase from Trametes versicolor. Laccase is a copper-containing enzyme which inhibition might prevent or reduce the activity of the plant pathogens that produce it in various biochemical processes. The kinetic and molecular modeling studies were performed and for selected compounds, the docking results were discussed. Seven 4-hydroxybenzhydrazide (4-HBAH) derivatives exhibited micromolar activity Ki = 24–674 µM with the predicted and desirable competitive type of inhibition. The structure–activity relationship (SAR) analysis revealed that a slim salicylic aldehyde framework had a pivotal role in stabilization of the molecules near the substrate docking site. Furthermore, the presence of phenyl and bulky tert-butyl substituents in position 3 in salicylic aldehyde fragment favored strong interaction with the substrate-binding pocket in laccase. Both 3- and 4-HBAH derivatives containing larger 3-tert-butyl-5-methyl- or 3,5-di-tert-butyl-2-hydroxy-benzylidene unit, did not bind to the active site of laccase and, interestingly, acted as non-competitive (Ki = 32.0 µM) or uncompetitive (Ki = 17.9 µM) inhibitors, respectively. From the easily available laccase inhibitors only sodium azide, harmful to environment and non-specific, was over 6 times more active than the above compounds.
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Olczak, Teresa, Dabney White Dixon, and Caroline Attardo Genco. "Binding Specificity of the Porphyromonas gingivalis Heme and Hemoglobin Receptor HmuR, Gingipain K, and Gingipain R1 for Heme, Porphyrins, and Metalloporphyrins." Journal of Bacteriology 183, no. 19 (October 1, 2001): 5599–608. http://dx.doi.org/10.1128/jb.183.19.5599-5608.2001.
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ABSTRACT Previous genetic and biochemical studies have confirmed that hemoglobin and hemin utilization in Porphyromonas gingivalis is mediated by the outer membrane hemoglobin and heme receptor HmuR, as well as gingipain K (Kgp), a lysine-specific cysteine protease, and gingipain R1 (HRgpA), one of two arginine-specific cysteine proteases. In this study we report on the binding specificity of the recombinant P. gingivalis HmuR protein and native gingipains for hemoglobin, hemin, various porphyrins, and metalloporphyrins as assessed by spectrophotometric assays, by affinity chromatography, and by enzyme-linked immunosorbent assay. Protoporphyrin, mesoporphyrin, deuteroporphyrin, hematoporphyrin, and some of their iron, copper, and zinc derivatives were examined to evaluate the role of both the central metal ion and the peripheral substituents on binding to recombinant HmuR and soluble gingipains. Scatchard analysis of hemin binding to Escherichia coli cells expressing recombinant membrane-associated six-His-tagged HmuR yielded a linear plot with a binding affinity of 2.4 × 10−5 M. RecombinantE. coli cells bound the iron, copper, and zinc derivatives of protoporphyrin IX (PPIX) with similar affinities, and approximately four times more tightly than PPIX itself, which suggests that the active site of HmuR contains a histidine that binds the metal ion in the porphyrin ring. Furthermore, we found that recombinant HmuR prefers the ethyl and vinyl side chains of the PPIX molecule to either the larger hydroxyethyl or smaller hydrogen side chains. Kgp and HRgpA were demonstrated to bind various porphyrins and metalloporphyrins with affinities similar to those for hemin, indicating that the binding of Kgp and HRgpA to these porphyrins does not require a metal within the porphyrin ring. We did not detect the binding of RgpB, the arginine-specific cysteine protease that lacks a C-terminal hemagglutinin domain, to hemoglobin, porphyrins, or metalloporphyrins. Kgp and HRgpA, but not RgpB, were demonstrated to bind directly to soluble recombinant six-His-tagged HmuR. Several possible mechanisms for the cooperation between outer membrane receptor HmuR and proteases Kgp and HRgpA in hemin and hemoglobin binding and utilization are discussed.
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Banci, Lucia, Ivano Bertini, Vito Calderone, Nunzia Della-Malva, Isabella C. Felli, Sara Neri, Anna Pavelkova, and Antonio Rosato. "Copper(I)-mediated protein–protein interactions result from suboptimal interaction surfaces." Biochemical Journal 422, no. 1 (July 29, 2009): 37–42. http://dx.doi.org/10.1042/bj20090422.
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The homoeostasis of metal ions in cells is the result of the contribution of several cellular pathways that involve transient, often weak, protein–protein interactions. Metal transfer typically implies the formation of adducts where the metal itself acts as a bridge between proteins, by co-ordinating residues of both interacting partners. In the present study we address the interaction between the human copper(I)-chaperone HAH1 (human ATX1 homologue) and a metal-binding domain in one of its partners, namely the P-type copper-transporting ATPase, ATP7A (ATPase, Cu+ transporting, α polypeptide). The adduct was structurally characterized in solution, in the presence of copper(I), and through X-ray crystallography, upon replacing copper(I) with cadmium(II). Further insight was obtained through molecular modelling techniques and site-directed mutagenesis. It was found that the interaction involves a relatively small interface (less than 1000 Å2, 1 Å=0.1 nm) with a low fraction of non-polar atoms. These observations provide a possible explanation for the low affinity of the two apoproteins. It appears that electrostatics is important in selecting which domain of the ATPase is able to form detectable amounts of the metal-mediated adduct with HAH1.
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Das, Dola, Nisha Tapryal, Shyamal K. Goswami, Paul L. Fox, and Chinmay K. Mukhopadhyay. "Regulation of ceruloplasmin in human hepatic cells by redox active copper: identification of a novel AP-1 site in the ceruloplasmin gene." Biochemical Journal 402, no. 1 (January 25, 2007): 135–41. http://dx.doi.org/10.1042/bj20060963.
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Cp (ceruloplasmin), a copper containing plasma protein, mainly synthesized in the liver, is known to be functional between the interface of iron and copper metabolism. We have reported previously that Cp is regulated by cellular iron status, but the process of the regulation of Cp by copper still remains a subject for investigation. In the present paper, we show that PDTC (pyrrolidine dithiocarbamate), a thiol compound widely known to increase intracellular redox copper, regulates Cp expression in hepatic cells by a copper-dependent transcriptional mechanism. To find out the mechanism of induction, chimeric constructs of the Cp 5′-flanking region driving luciferase were transfected into human hepatic cells. Deletion and mutational analyses showed the requirement of a novel APRE [AP-1 (activator protein-1) responsive element] present about 3.7 kb upstream of the translation initiation site. The role of AP-1 was confirmed by electrophoretic mobility-shift analysis. Western blot and overexpression studies detected the AP-1 as a heterodimer of c-jun and c-fos proteins. The activation of AP-1 was found to be copper-dependent as a specific extracellular chelator bathocuproine disulfonic acid blocked PDTC-mediated induction of AP-1–DNA binding and increased reporter gene activity. Whereas, in a copper-free medium, PDTC failed to activate either AP-1 or Cp synthesis, supplementation of copper could reverse AP-1 activation and Cp synthesis. Our finding is not only the first demonstration of regulation of Cp by redox copper but may also explain previous findings of increased Cp expression in cancers like hepatocarcinoma, where the intracellular copper level is higher in a redox compromised environment.
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Urresti, Saioa, Alan Cartmell, Feng Liu, Paul H. Walton, and Gideon J. Davies. "Structural studies of the unusual metal-ion site of the GH124 endoglucanase from Ruminiclostridium thermocellum." Acta Crystallographica Section F Structural Biology Communications 74, no. 8 (August 1, 2018): 496–505. http://dx.doi.org/10.1107/s2053230x18006842.
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The recent discovery of `lytic' polysaccharide monooxygenases, copper-dependent enzymes for biomass degradation, has provided new impetus for the analysis of unusual metal-ion sites in carbohydrate-active enzymes. In this context, the CAZY family GH124 endoglucanase from Ruminiclostridium thermocellum contains an unusual metal-ion site, which was originally modelled as a Ca2+ site but features aspartic acid, asparagine and two histidine imidazoles as coordinating residues, which are more consistent with a transition-metal binding environment. It was sought to analyse whether the GH124 metal-ion site might accommodate other metals. It is demonstrated through thermal unfolding experiments that this metal-ion site can accommodate a range of transition metals (Fe2+, Cu2+, Mn2+ and Ni2+), whilst the three-dimensional structure and mass spectrometry show that one of the histidines is partially covalently modified and is present as a 2-oxohistidine residue; a feature that is rarely observed but that is believed to be involved in an `off-switch' to transition-metal binding. Atomic resolution (<1.1 Å) complexes define the metal-ion site and also reveal the binding of an unusual fructosylated oligosaccharide, which was presumably present as a contaminant in the cellohexaose used for crystallization. Although it has not been possible to detect a biological role for the unusual metal-ion site, this work highlights the need to study some of the many metal-ion sites in carbohydrate-active enzymes that have long been overlooked or previously mis-assigned.
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Varela-Nallar, Lorena, Enrique M. Toledo, Luis F. Larrondo, Ana L. B. Cabral, Vilma R. Martins, and Nibaldo C. Inestrosa. "Induction of cellular prion protein gene expression by copper in neurons." American Journal of Physiology-Cell Physiology 290, no. 1 (January 2006): C271—C281. http://dx.doi.org/10.1152/ajpcell.00160.2005.
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Prion diseases are caused by the conformational transition of the native α-helical cellular prion protein (PrPC) into a β-sheet pathogenic isoform. However, the normal physiological function of PrPC remains elusive. We report herein that copper induces PrPC expression in primary hippocampal and cortical neurons. PrPC induced by copper has a normal glycosylation pattern, is proteinase K-sensitive and reaches the cell surface attached by a glycosyl phosphatidylinositol anchor. Immunofluorescence analysis revealed that copper induces PrPC levels in the cell surface and in an intracellular compartment that we identified as the Golgi complex. In addition, copper induced the activity of a reporter vector driven by the rat PrPC gene ( Prnp) promoter stably transfected into PC12 cells, whereas no effect was observed in glial C6 clones. Also cadmium, but not zinc or manganese, upregulated Prnp promoter activity in PC12 clones. Progressive deletions of the promoter revealed that the region essential for copper modulation contains a putative metal responsive element. Although electrophoretic mobility shift assay demonstrated nuclear protein binding to this element, supershift analysis showed that this is not a binding site for the metal responsive transcription factor-1 (MTF-1). The MTF-1-independent transcriptional activation of Prnp is supported by the lack of Prnp promoter activation by zinc. These findings demonstrate that Prnp expression is upregulated by copper in neuronal cells by an MTF-1-independent mechanism, and suggest a metal-specific modulation of Prnp in neurons.
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Huo, Chunheng, Tinghong Ming, Yan Wu, Hengshang Huan, Xiaoting Qiu, Chenyang Lu, Ye Li, Zhen Zhang, Jiaojiao Han, and Xiurong Su. "Structural and Biochemical Characterization of Silver/Copper Binding by Dendrorhynchus zhejiangensis Ferritin." Polymers 15, no. 5 (March 3, 2023): 1297. http://dx.doi.org/10.3390/polym15051297.
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Ferritin with a highly symmetrical cage-like structure is not only key in the reversible storage of iron in efficient ferroxidase activity; it also provides unique coordination environments for the conjugation of heavy metal ions other than those associated with iron. However, research regarding the effect of these bound heavy metal ions on ferritin is scarce. In the present study, we prepared a marine invertebrate ferritin from Dendrorhynchus zhejiangensis (DzFer) and found that it could withstand extreme pH fluctuation. We then demonstrated its capacity to interact with Ag+ or Cu2+ ions using various biochemical and spectroscopic methods and X-ray crystallography. Structural and biochemical analyses revealed that both Ag+ and Cu2+ were able to bind to the DzFer cage via metal-coordination bonds and that their binding sites were mainly located inside the three-fold channel of DzFer. Furthermore, Ag+ was shown to have a higher selectivity for sulfur-containing amino acid residues and appeared to bind preferentially at the ferroxidase site of DzFer as compared with Cu2+. Thus, it is far more likely to inhibit the ferroxidase activity of DzFer. The results provide new insights into the effect of heavy metal ions on the iron-binding capacity of a marine invertebrate ferritin.
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Goumakos, William, Jean-Pierre Laussac, and Bibudhendra Sarkar. "Binding of cadmium(II) and zinc(II) to human and dog serum albumins. An equilibrium dialysis and 113Cd-NMR study." Biochemistry and Cell Biology 69, no. 12 (December 1, 1991): 809–20. http://dx.doi.org/10.1139/o91-121.
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The binding of Cd(II) and Zn(II) to human serum albumin (HSA) and dog serum albumin (DSA) has been studied by equilibrium dialysis and 113Cd(II)-NMR techniques at physiological pH. Scatchard analysis of the equilibrium dialysis data indicate the presence of at least two classes of binding sites for Cd(II) and Zn(II). On analysis of the high-affinity class of sites, HSA is shown to bind 2.08 ± 0.09 (log K = 5.3 ± 0.6) and 1.07 ± 0.12 (log K = 6.4 ± 0.8) moles of Cd(II) and Zn(II) per mole of protein, respectively. DSA bound 2.02 ± 0.19 (log K = 5.1 ± 0.8), and 1.06 ± 0.15 (log K = 6.0 ± 0.2) moles of Cd(II) and Zn(II) per mole of protein, respectively. Competition studies indicate the presence of one high-affinity Cd(II) site on both HSA and DSA that is not affected by Zn(II) or Cu(II), and one high-affinity Zn(II) site on both HSA and DSA that is not affected by Cd(II) or Cu(II). 113Cadmium-HSA spectra display three resonances corresponding to three different sites of complexation. In site I, Cd(II) is most probably coordinated to two or three histidyl residues, site II to one histidyl residue and three oxygen ligands (carboxylate), while for the most upfield site III, four oxygens are likely to be involved in the binding of the metal ion. The 113Cd(II)-DSA spectra display only two resonances corresponding to two different sites of complexation. The environment around Cd(II) at sites I and II on DSA is similar to sites I and II, respectively, on HSA. No additional resonances are observed in any of these experiments and in particular in the low field region where sulfur coordination occurs. Overall, our results are consistent with the proposal that the physiologically important high-affinity Zn(II) and Cd(II) binding sites of albumins are located not at the Cu(II)-specific NH2-terminal site, but at internal sites, involving mostly nitrogen and oxygen ligands and no sulphur ligand.Key words: albumin, human serum, dog serum, cadmium, zinc, copper, NMR, equilibrium dialysis, binding.